Optical scanning microscope with linear scanning

ABSTRACT

Optical scanning microscope with linear scanning and with illumination of a specimen via a beam splitter, which is arranged in an objective pupil and consists of at least a reflecting first portion and at least a transmitting second portion, whereby the reflecting portion serves to couple in the illumination light and the transmitting portion serves to pass the detection light in the detection direction or the transmitting portion serves to couple in the illumination light and the reflecting portion serves to couple out the detection light, with a first scanning arrangement, whereby means are provided in the detection light path for the overlay of at least one further scanning arrangement for illumination and detection.

In DE10257237 A1 among other things a beam splitter is described for aline scanner.

In a line scanner the specimen is illuminated with a line focus (e.g.along the X-coordinate), which is shifted in the coordinate (Y)perpendicular to the line. For this the source of light is linearlyfocused into an intermediate image plane of the microscope mechanism bymeans of optics. By the focusing in Y direction in the intermediateimage, for example by a cylinder lens, a linear and diffraction-limiteddistribution of intensity arises along X on the specimen. With furtheroptics the light is focused into the pupil of the microscopearrangement. In the pupil levels of the microscope arrangement a linefocus results in each case. The pupil levels and the scanner areconjugate to each other and to the rear focal plane of the microscopearrangement, so that the scanner can induce the linear anddiffraction-limited focused distribution of intensity perpendicular tothis (Y-coordinate in the specimen). The focusing into the specimen ismade by scan optics, the tube lens and the objective. A relay opticsproduces conjugate pupil levels of the microscope arrangement. Due tothe kind of the specimen reciprocal effect e.g. during an excitation forfluorescence or luminescence the light emitted from the specimen is ofsmall spatial coherency. That is each point excited in the specimenradiates essentially independently of the neighboring points as pointemitter into all directions in space. The optics, (e.g. a microscopeobjective) displays the individual point emitters together with the tubelens TL into an intermediate image plane ZB of the microscope mechanism,whereby the pupil P is illuminated homogeneously (broken light path) bywave fronts that are essentially incoherent to each other and ofdifferent directions of propagation. In the pupil is the element whichseparates the excitation light from the detection light. It isconstructed as described in DE.

FIG. 1:

The light of a far field-source of light LQ is focused with suitableoptics for the production of an illumination line, for example acylinder lens ZL, linearly into one level that is conjugate to the pupilP of the microscope objective O, in which there is a developed beamsplitter ST in accordance with DE 10257237A1, which exhibits a narrowlinear transmitter range, over which the line is displayed viatransmission optics L1, L2, scan optics SO, tube lens TL and objective Ointo the specimen PR. A scanner SC is arranged in a pupil P, that movesthe line quickly over the specimen in a scan direction perpendicular tothe line expansion. The light (broken) coming from the specimen, isreturned by the beam splitter reflecting up to the narrow transmitterrange in direction of detection via a replaceable filter F as well asdetection optics PO toward a detector DE1, in front of which a slitdiaphragm can be arranged.

FIG. 2:

Here exemplary sources of light LQ1, LQ2 are represented in addition tothe elements represented in FIG. 1 on cross ports, which can result alsofrom bypass of only one source of light, whereby wavelength andintensity can be adjusted advantageously.

By use of achromatic beam splitters as described in DE . . . the specialadvantage is that the same wavelength can be used for both sources oflight LQ1, LQ2, which can be formed also by allocation in and of thesame source of light. The intermediate images ZB and ZB1 are levelsconjugate to each other. Furthermore, the pupil levels of the microscopearrangement P are conjugate levels to each other. The conjugate levelsin each case are produced by the effect of the optics lying between themin each case (those acting as relay optics-light paths onlyschematically drawn).

LQ 2 can be for example a point scanner. The illumination light of thepoint scanner can be used advantageously for the purposeful manipulation(e.g. uncaging) on certain specimen ranges.

The illumination light of LQ2 is faded after passage by separate scanoptics SO2 as well as a scanner SC1 (a X/Y scanner favorable) over ausual dichroic color divider FT1 into the detection light path of theline scanner and arrives over the reflective range of the divider STtoward the specimen PR.

The reflecting range of the beam splitter ST is thus used advantageouslyfor the reflection of a further scan light path.

The light coming from the specimen arrives on the one hand at thedetector DE1 and on the other hand depending on interpretation of thecolor divider FT1 also via a further color divider FT2 toward a seconddetector DE2.

For example fluorescence light excited by LQ1 coming from the specimenarrives during appropriate interpretation by FT1 on the detector DE1while reflected light of the point scanner LQ2 arrives on the detectorDE2. Furthermore different fluorescence wavelengths excited also by LQland LQ2 can arrive on the different detectors DE1 and DE2.

Since the light moved by the scanner SC1 is moved here additionally bythe scanner 1, the scanner SC1 must be controlled in such a way that itcompensates for the movement of the scanner SC and additionally realizesa relative position for line illumination.

That is simple to realize if scanner SC1 moves slower in comparison tothe scanner SC2.

The fluorescence light induced by LQ2 can be also guided on the linedetector DE1.

Depending on the position of the scanner 2 the fluorescent spot movesaway over the line detector DE1, i.e. the light is separated by thescanner 2 toward DE1.

FIG. 3

Here a cross port KS1 is provided, that can be a separate module and isbetween a microscope stand S with tube lens and objective, a first scanunit SC1 and a second scan unit SC2.

SC1 can contain the described line scanner and SC2 a point scanner forscanning and/or manipulation. SC1 and SC2 are couplable with KS1 atinterfaces.

For this several intermediate images ZB that are conjugate to each otherare available in KS1 (via the optics L1, L2). The conjugate levels ineach case are produced by the effect of the optics lying between them(light paths only schematic).

At the beam splitter ST1, which is developed analogous to the beamsplitter ST (DE . . .) a line is focused on the specimen by thetransmitting range. It is attached in one pupil level of the microscopearrangement.

For example with SC1 excited light such as fluorescence light in thespecimen is reflected downward at ST1 and arrives over FT3, which ishere constructed such that it lets this light portion pass through, aswell as over several reflectors RF onto the other side of ST1. Thislight is diverted by ST1 toward the detector DE1 via ST.

The fluorescence light excited by the line scanner, which is reflectedat ST1 to the side, is thus brought advantageously in the entire widthback into the light path toward DE1.

Thus a further scanner SC2 can be reflected via FT3, whereby byappropriate training of FT3, which can be replaceable, differentfluorescence wavelengths can arrive at DE1 and/or DE2. The mode ofoperation is similar to the one described above.

Contrary to FIG. 3 the scanners 1 and 2 can work here advantageouslyindependent of each other.

FIG. 4:

Here the light is not guided via reflectors RF on the back side of thebeam splitter ST1 as in FIG. 4 but on the back side of the scanner SC33,which is here a mirror that can reflect on its front and back sides andfurther guides with its back side the specimen light (descanned) comingfrom the specimen and excited by the line scanner (LQ1 arrives fromabove on the front side of the scanner mirror) to the detector DE1. FT3is constructed here in such a way that it lets through the lightintended for the detector DE1 and only reflects the light intended forDE2.

Thereby again different fluorescences excited by the line scanner andthe point scanner can be detected advantageously at the same time.

FIG. 5:

Here the light excited by the line scanner is not descanned as in FIG. 5but arrives via FT1 directly at a surface detector (CCD matrix, gegatetecamera), i.e. the linear light distribution coming from the specimenruns in the direction of the scan via the receiver surfaces, whichrecords thereby a specimen image.

Further scan arrangements can also be reflected by cascading(arrangement of further color dividers FT into a common light path). Thescan arrangements can be arbitrary image-giving arrangements. Examplesare the already mentioned point scanners, scanners of point ofresonance, Nipkow scanner, line scanners and multi-point scanners.Furthermore, these can also be far-field based microscope systems. It isadvantageous here that they exhibit an intermediate image plane asinterface.

1-4. (canceled)
 5. An optical scanning microscope with linear scanning,comprising: a source of illumination light, an objective pupil, a beamsplitter for illuminating a specimen, the beam splitter being arrangedin the objective pupil and including at least one reflecting firstportion and at least one transmitting second portion, wherein one of thefirst and second portion couples in the illumination light and the otherof the first and second portion passes detection light in a detectiondirection, a first scanning arrangement for line scanning ofillumination light from the line forming means over the specimen, and atleast one further scanning arrangement for illumination and detection,provided in the detection light path.
 6. An optical scanning microscopewith linear scanning, comprising: a source of illumination light, anobjective pupil, a beam splitter for illuminating a specimen, the beamsplitter being arranged in the objective pupil and including at leastone reflecting first portion and at least one transmitting secondportion, wherein one of the first and second portions couples in theillumination light and the other of the first and second portion passesdetection light in a detection direction, and wherein the beam splitterhas a side facing towards the specimen and a side facing away from thespecimen, a first scanning arrangement for linear scanning ofillumination light from the line forming means over the specimen, adetection unit for detecting specimen light from the first scanningarrangement, at least one further scanning arrangement coupled in viathe beam splitter, and returning means for detouring of a part of thespecimen light at the side of the beam splitter facing away from thespecimen and for then returning the detoured specimen light toward thedetection unit for detecting specimen light from the first scanningarrangement.
 7. An optical scanning microscope with linear scanning,comprising: a source of illumination light, an objective pupil, a beamsplitter for illuminating a specimen, the beam splitter being arrangedin the objective pupil and including at least one reflecting firstportion and at least one transmitting second portion, wherein one of thefirst and second portion couples in the illumination light and the otherof the first and second portion passes detection light in a detectiondirection, a first scanning arrangement for linear scanning ofillumination light from the line forming means over the specimen, thefirst scanning arrangement having at least one scan mirror, the scanmirror having a reflecting rear side, at least one further scanningarrangement coupled in via the beam splitter, and returning means fordirecting at least one part of the specimen light to the reflecting rearside of the scan mirror for return in the detection direction.
 8. Anoptical scanning microscope with linear scanning, comprising: a sourceof illumination light, an objective pupil, a beam splitter forilluminating a specimen, the beam splitter being arranged in theobjective pupil and including at least one reflecting first portion andat least one transmitting second portion, wherein one of the first andsecond portion couples in the illumination light and the other of thefirst and second portion passes detection light in a detectiondirection, a first scanning arrangement for linear scanning ofillumination light from the line forming means over the specimen, atleast one further scanning arrangement coupled in via the beam splitter,and means for detecting specimen light coming from the beam splitterwithout descanning.